Total RNA was extracted from all cell lines using the TRIzol® RNA purification reagent. RNA quantity and purity were determined by using a NanoDrop ND-1000. Total RNA (1 µg) from each sample was reverse transcribed and real-time RT–PCR measurements were performed as described previously [9] (link) using an Mx3000P apparatus (Agilent) with the corresponding SYBR Green kit. PCR primers were designed with Primer 3 (Agilent). Gene expression was normalized to TBP and RPLP0 (also known as 36B4).
Mx3000p apparatus
Manufactured by Agilent Technologies
Sourced in United States
The Mx3000P is a real-time PCR (qPCR) system designed for gene expression analysis and nucleic acid quantification. It features a compact design, intuitive software, and a range of sample block options to accommodate various sample volumes and formats. The Mx3000P provides precise temperature control, sensitive optical detection, and robust data analysis capabilities to support a variety of real-time PCR applications.
Automatically generated - may contain errors
4 protocols using mx3000p apparatus
1
RNA Extraction and qRT-PCR Analysis
2
Quantitative Gene Expression Analysis
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Total RNA was extracted from all cell lines using the TRIzol® RNA purification reagent. RNA quantity and purity were determined by using a NanoDrop ND-1000. Total RNA (1 μg) from each sample was reverse transcribed and real-time RT–PCR measurements were performed as described previously47 (link) using an Mx3000 P apparatus (Agilent, CA, USA) with the corresponding SYBR Green kit. PCR primers were designed with Primer 3 (Agilent, CA, USA). Gene expression was normalized to the TATA box Binding Protein (TBP) and GAPDH (GlycerAldehyde-3-Phosphate DeHydrogenase). The sequence of primers used is indicated in Table S1 .
3
Extraction and Quantification of Total RNA
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Total RNA was extracted from cell or tumors using the TRIzol RNA purification reagent (Invitrogen, Waltham, MA, USA). The purification of RNA from the tumors was performed using TissueLyser II (Qiagen, Hilden, Germany). Briefly, the samples were placed on dry ice, and two TissueLyser beads (3 mm, Qiagen, Hilden, Germany) and 1 mL of TRIzol were added. The samples were then shaken twice for 2 min at 30 Hz.
RNA quantity and purity were determined using a spectrophotometer (DeNovix Inc., Wilmington, DE, USA). Total RNA (1 μg) from each sample was reverse-transcribed using hexanucleotides. Real-time RT–PCR measurements were performed with an Mx3000P apparatus (Agilent Technologies, Santa Clara, CA, USA) with the corresponding SYBR Green kit. PCR primers were designed with the Primer Blast or Primer BD programs and obtained from Promega, Madisson, WI, USA. Gene expression was normalized to β-actin. All primers used are indicated inSupplementary Materials Table S1 .
RNA quantity and purity were determined using a spectrophotometer (DeNovix Inc., Wilmington, DE, USA). Total RNA (1 μg) from each sample was reverse-transcribed using hexanucleotides. Real-time RT–PCR measurements were performed with an Mx3000P apparatus (Agilent Technologies, Santa Clara, CA, USA) with the corresponding SYBR Green kit. PCR primers were designed with the Primer Blast or Primer BD programs and obtained from Promega, Madisson, WI, USA. Gene expression was normalized to β-actin. All primers used are indicated in
4
Quantifying VEGF Expression in CRC Cells
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Total RNA was extracted from CRC cells using the TRIzol® RNA purification reagent. RNA quantity and purity were determined by using a NanoDrop ND-1000. Total RNA (1 μg) from each sample was reverse transcribed and real-time RT-PCR measurements were performed as described previously [40 (link), 41 (link)] using a Mx3000P apparatus (Agilent) with the corresponding SYBR Green kit. PCR primers were designed with Primer 3 (Agilent) as follows: VEGF, upper, 5’-CGAAGTGGTGAAGTTCATGGATG-3’, lower, 5’-TTCTGTATCAGTC TTTCCTGGTGAG. 36B4 (also known as RPLP0), upper, 5’-GATTGGCTACCCAACTGTTG-3’; lower, 5’-CAGGGGCAGCAGCCAC AAA-3’. Gene expression was normalized to 36B4.
Western blot analysis was carried out as described previously [42 (link)]. The primary antibodies were directed against HIF-1alpha (BD Bioscience # 610958), HIF-2alpha (Novus Biological # NB-100122) or beta-actin (Santa Cruz # sc-47778), while the corresponding secondary antibodies were purchased from Jackson ImmunoResearch.
Western blot analysis was carried out as described previously [42 (link)]. The primary antibodies were directed against HIF-1alpha (BD Bioscience # 610958), HIF-2alpha (Novus Biological # NB-100122) or beta-actin (Santa Cruz # sc-47778), while the corresponding secondary antibodies were purchased from Jackson ImmunoResearch.
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